Lakshmi Kantham T et al / Int. J. Res. Ayurveda Pharm. 8 (Suppl 2), Research Article.
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1 Research Article ACUTE, SUB-ACUTE (8-DAYS), AND SUB-CHRONIC (90-DAYS) ORAL TOXICITY STUDIES OF NANDHI MEZHUGU Lakshmi Kantham T 1 *, Ganapathy G, Suba V, Srinivasan M. R, Geetha A 5 Lecturer, Department of Maruthuvam, National Institute of Siddha, Chennai, Tamilnadu, India Former Professor, Department of Kuzhandhai maruthuvam, National Institute of Siddha, Chennai, India Assistant Professor, Department of Pharmacology, National Institute of Siddha, Chennai, India Assistant Professor, Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai, India 5 Senior Research Fellow, Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai, India *Corresponding author drlakshmiramaswamy@gmail.com DOI: 1897/ ABSTRACT Received on: 8/0/17 Accepted on: 19/0/17 Nandhi Mezhugu (NM) is a classical Siddha herbo-mineral formulation indicated for all types of Arthritides. Acute toxicity study was performed at dose levels, i.e., 50, 00, and 000 mg/kg body weight of rats. Sub-acute toxicity study was performed at doses of 9, 5, body weight and sub-chronic toxicity study was performed at doses of 9, 5, body weight of rats. Palm Jaggery solution was used as vehicle. In sub-acute and sub-chronic toxicity studies, the drug was administered twice for 7 days followed by 7 days of drug holiday, i.e., in sub-acute toxicity study, the period was 56 days and in the sub-chronic toxicity study, it was 180 days. In both studies, the high dose recovery and recovery control groups were used to study the safety of the drug. Biochemical, haematological parameters, gross pathology and histopathology of the rats were analysed in both studies. ICP-OES detection of heavy metal traces in animal tissue samples in sub-chronic toxicity study (high dose group) were analysed. Acute toxicity study showed no mortality and no treatment-related toxicity signs. Similarly, sub-acute and sub-chronic toxicity studies showed no treatment-related abnormalities at the high dose levels such as 90 and body weight in rats respectively. Hence, the scientific validation of the safety of Nandhimezhugu is proven by the toxicity studies. Keywords: Nandhimezhugu, Mercury, Arsenic, Seraankottai, Toxicitystudies, OECD guidelines. INTRODUCTION Siddha system of medicine is a comprehensive ancient system having its roots in the Tamil- speaking land of South India. This system was founded by Siddhars who expounded the philosophy and theory of immortality. This system employs a holistic approach in its treatment methodology and has made enormous contributions towards the health care of Tamil people. The siddha system of medicine is based on the resources derived from herbs, metals, minerals and animal products. For global acceptance, this system of medicine should undergo scientific validation, i.e., upgrading the levels of quality, safety, reliability and efficacy. The siddha herbo-mineral formulation, Nandhi Mezhugu (NM), is one of the versatile classical siddha medicines recommended for a wide range of indications by siddha practitioners 1,. It is the need of the hour to do scientific validation related to quality and safety for this drug to enter the global platform. MATERIALS AND METHODS Preparation of Nandhi Mezhugu Preparation as per The Siddha formulary of India Part 1 and Siddha Vaidhiya Thirattu 1,, Details of Trail Drug Nandhi Mezhugu Batch No: S Mfg Date: Exp Date: 5 years from the date of manufacture. Manufacturing Unit: IMPCOPS (Indian Medicine Practitioners Co-operative Society) Pharmacy, Chennai-1. Animal Care and Husbandry The study protocol involving animals was reviewed and accepted by Institutional Animal Ethical Committee (IAEC), National Institute of Siddha, Chennai-60007, with the experimental protocol IAEC approved Number: 18/AC/09/CPCSEA- /June011/10 for acute toxicity study and 18/AC/09/CPCSEA-9/Dec01/5 for sub-acute and subchronic toxicity studies. Experiments were performed as per the guidelines prescribed by the Committee for the purpose of conduct and supervisions of experiments on animals (CPCSEA). Male and female Wistar albino rats ( g) were obtained from Laboratory Animal Medicine, Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai and housed in Animal House of National Institute of Siddha. Each group of rats was separately housed in polypropylene cages in a wellventilated room under an ambient temperature of C and 5-75 relative humidity, with a 1-h light/dark artificial light cycle. They were provided with rodent chow from VRK Nutritional Solutions, Sangli, Maharastra and reverse osmosis purified water ad libitum. All the animals were acclimatized to the laboratory conditions for 7 days, prior to experimentation. 18
2 Acute Toxicity Studies The acute oral toxicity study was performed in accordance with OECD test guideline. The limit test dose of 000 mg/kg b.w of rat was used as stipulated in OECD guidelines. The test drug suspension was administered at three dose levels such as 50, 00 and 000 mg/kg body weight once orally to the fasted rats. The following observations were made in all the dose level groups. a. Mortality and morbidity were noted. b. Following the trail drug administration, weekly body weight was recorded. c. Clinical signs such as lethality, convulsion, tremor, straub tail, motor coordination, abnormal gait (tiptoe, rolling), fore paw treading, sedation, excitation, jumps, loss of balance, writhes, piloerection, stereotypies (head movements, chewing), head twitches, scratching, respiration, aggressiveness, fear, reactivity to touch, muscle tone, loss of righting reflex, ptosis, exophthalmos, loss of grasping, akinesia, catalepsy, loss of traction, loss of corneal reflex, analgesia, lacrimation, salivation, defecation and other behavioural changes were observed at about 0 mins, 1 hr, hr and hr on day 1 and daily thereafter for 1 days. At the end of 1 th day, the experimental animals were necropsied and investigated for gross pathological examination. Sub-acute Toxicity Study Justification for Dose Selection As per the result of acute toxicity study in Wistar rats, it was inferred that NM was non-toxic up to the maximum dose of 000 mg/kg b.w. On the basis of these results and the therapeutic dose for humans as mentioned in Siddha Literature, which was extrapolated to the body surface area of rat, the following doses of, and b.w were selected for the sub-acute toxicity study and administered twice daily by oral route. Preparation and Administration of Dose Repeated-dose (8 days) oral toxicity study was carried out according to OECD guideline The animals were divided into six groups of 10 animals each (5 males and 5 females). Group 1 received Palm Jaggery solution and considered as Vehicle Control. Groups II, III and IV (, Mid Dose, and High Dose respectively) received test drug suspensions, (NM with Palm Jaggery solution) at doses of 9, 5 and b.w respectively. Group V (Recovery control) and Group VI (High dose recovery). The NM was administered by oral gavage for 8 days ( weeks) of dosing duration at regular intervals twice daily as per the protocol mentioned in the Siddha literature where 7 days of test drug administration is followed by 7 days of drug holiday alternatively for four cycles of dosing to match 8 days of dosing period. Recovery groups were scheduled for follow-up observations for the next 1 days without NM administration. recovery and recovery control were included in the study to determine the delayed occurrence, or persistence of, or recovery from toxic effects, if any. All groups were observed twice daily for morbidity and mortality, and clinical signs, once daily. The bodyweight and food intake of the animals were evaluated at weekly intervals. Any change in water consumption was observed and recorded. On the 57 th day, after an overnight fasting, the rats were anaesthetized with Thiopentone sodium and blood samples were collected from retro-orbital plexus under light ether anaesthesia by trained personnel. The blood samples for haematological and biochemical analysis were collected in tubes with and without EDTA, respectively. Total WBC, Differential Counts, RBC, Haemoglobin, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, PDW and PCT were analysed. Biochemical analysis was performed on serum obtained after centrifugation of total blood (without EDTA) at 500 rpm for 15 min. Blood Glucose, Urea, Creatinine, Cholesterol, Triglyceride, HDL, LDL, Total serum protein, ALT or SGPT, AST or SGOT were analysed. Sub Chronic Toxicity Study Justification for Dose Selection As stated in results of acute toxicity studies in Wistar rats indicated that NM was nontoxic up to the maximum dose of 000 mg/kg b.w. On the basis of these results and the therapeutic dose as mentioned in Siddha Literature, which was extrapolated to body surface area of rat, the following doses of, and b.w were selected for the study and administered orally twice daily. Preparation and Administration of Dose Repeated-dose oral toxicity study was carried out according to OECD test guideline The animals were divided into six groups of 10 animals each (5 males and 5 females). Group I received Palm Jaggery solution and considered as Vehicle control. Groups II, III and IV (Low Dose, Mid Dose, and High Dose respectively) received test drug suspensions, (NM with Palm Jaggery solution) at doses 9, 5 and b.w respectively. Group V (Recovery control) and Group VI (High dose recovery). The NM was administered twice daily by oral gavage for 7 days followed by 7 days drug holiday alternatively for 180 days (90 days dosing and 90 days drug holiday). This protocol is followed to mimic the treatment protocol in humans as mentioned in the traditional Siddha Literature. Recovery groups were scheduled for follow-up observations for the next 1 days without NM administration. recovery and recovery control were included in the recovery study to determine the delayed occurrence, or persistence of, or recovery from toxic effects. All groups were observed twice daily for morbidity and mortality, and clinical signs, once daily. Bodyweight and food intake of the animals were evaluated weekly. Any change in the water consumption was observed and recorded. On the 181 st day, after an overnight fasting, the rats were anaesthetized with Thiopentone sodium and blood sample for haematological and biochemical analysis were collected into tubes with and without EDTA, respectively. Total WBC, Differential Counts, RBC, Haemoglobin, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, PDW and PCT. Biochemical analysis was performed on serum obtained after centrifugation of total blood (without anticoagulant) at 500 rpm for 15 min. Blood Glucose, Urea, Creatinine, Cholesterol, Triglyceride, HDL, LDL, Total serum protein, ALT or SGPT, AST or SGOT were screened. Histopathology In subacute toxicity study, necropsy was done in all animals in Group I to Group IV on 57 th day and Group V and Group VI (recovery groups) were done on 70 th day. In sub chronic toxicity study, necropsy was done in all animals in Group I to IV on 181 st day and the necropsy for Group V and Group VI (recovery groups) were performed on 195 th day. Blood samples were collected from abdominal aorta following which all the animals were euthanized by excess dose of Thiopentone sodium administered through intraperitoneal route. The gross pathological examinations of all major internal organs were 18
3 performed. The organs such as brain, heart, lungs, liver, kidneys, and spleen were weighed and the relative weights of the organs were calculated as mentioned in both studies. The organs were fixed in 10 neutral buffered formalin, trimmed and 5μ thickness of tissue sections were stained with hematoxylin and eosin for histopathological investigation. if the number of groups to be compared is more than two followed by Post-hoc test, Dunnet-t test. If only two groups are to be compared as in the case of recovery groups, Student s t-test was used. It was assumed that the data were normally distributed and there was a homogeneity of variance. P value < 0.05 was considered as statistically significant. Statistical Analysis: All the values are expressed as Mean SEM. The data were statistically analyzed by one-way ANOVA Table 1: Clinical signs or behavioural observations of the rat in the acute toxicity study S.No Dose mg/kg Control NM (50mg/kg) NM (00mg/kg) NM (000mg/kg) (+ indicates that the particular behaviour is present and indicates absence of the behaviour in animals) 1.Alertness. Aggressiveness. Piloerection. Grooming 5. Gripping 6. Touch Response 7. Decreased Motor Activity 8. Tremors 9. Convulsions 10. Muscle Spasm 11. Catatonia 1. Muscle relaxant 1. Hypnosis 1. Analgesia 15. Lacrimation 16.Exophthalmos 17.Diarrhoea 18.Writhing 19. Abnormal Respiration 0. Mortality. Table : Effect of Nandhi mezhugu on hematological parameters of wistar rats in sub-acute toxicity study Groups WBC Mon Gran Mono Gran RBC 10 1 /L HGB Vehicle control ** ** ** 1.0 ** 8.91 ** 0.5 ** ** * ** All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Recovery Groups Groups WBC Mon Gran Mono Gran RBC 10 1 /L HGB Recovery Control recovery All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Groups HCT MCV MCH pg MCHC RDW PLT MPV Fl PDW Fl PCT Vehicle control * * * * * All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p <
4 Recovery Groups Groups HCT MCV MCH pg MCHC RDW PLT MPV PDW fl PCT Recovery Control recovery All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Table : Effect of Nandhi mezhugu on biochemical parameters of experimental wistar rats in sub-acute toxicity study Groups Glucose Urea Creatinine Cholesterol TGL HDL LDL Vehicle control ***. 1.1* * All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Recovery Groups Groups Glucose Control Recovery Recovery Urea Creatinine Cholesterol TGL HDL LDL Table : Effect of Nandhi mezhugu on liver markers of experimental wistar rats in sub-acute toxicity study Groups TRP ALB SBIL SGOT SGPT SAP Vehicle control b.w * All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Recovery group Groups TRP ALB SBIL SGOT SGPT SAP Control Recovery Recovery All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p <
5 Table 5: Effect of nandhi mezhugu on hematological parameters of experimental wistar rats in sub-chronic oral toxicity study Groups WBC Mon Gran Mono Gran RBC 10 1 /L HGB Vehicle control ** ** ** All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Recovery Group Groups WBC Mon Gran Mono Gran RBC 10 1 /L HGB Recovery Control recovery All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Groups HCT MCV MCH pg MCHC RDW PLT MPV PDW PCT Vehicle control mg/kg All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Recovery Group Groups HCT MCV MCH pg MCHC RDW PLT MPV fl PDW fl PCT Recovery Control recovery All values are mean SEM. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Table 6: Effect of Nandhi mezhugu on serum biochemical parameters of experimental wistar rats sub-chronic oral toxicity study Groups Glucose Urea Creatinine Cholesterol TGL HDL LDL Vehicle control * * * * All values are mean SE. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p <
6 Recovery Group Groups Glucose Urea Creatinine Cholesterol TGL HDL LDL Recovery Control Recovery All values are mean SE. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Table 7: Effect of Nandhi mezhugu on liver markers of experimental wistar rats sub-chronic oral toxicity study Groups TRP ALB SBIL SGOT SGPT SAP Vehicle control ** ** ** All values are mean SE. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Groups TRP ALB SBIL SGOT SGPT SAP Recovery Control Recovery All values are mean SE. Statistical analysis by one-way ANOVA followed by Dunnett s Multiple Comparison. **p < *p < 0.01 Figure: 1 weekly body weight of rat in sub-acute toxicity study Figure: weekly body weight of rat in sub-chronic toxicity study RESULTS AND DISCUSSION Acute Toxicity Study The limit dose of 000 mg/kg did not cause mortality or any signs of acute toxicity, in the three rats on single dosing by oral route, for a short period (8 h) and long period (1 days). No mortality or behavioural changes (see Table-1) were observed at the end of the treatment. Similarly, subsequent study with 50, 00 and 000 mg/kg did not show any significant differences in bodyweight between control and treated groups during this period. It was observed that there was no mortality and toxic signs up to 000 mg/kg. NM can be classified under category-5 since the LD50 7 value was greater than 000 mg/kg in accordance with GHS [Globally Harmonized System of Classification and Labelling of Chemicals] 8 and this provides us a direct relevance for protecting human and animal health. Therefore, it can be concluded that Nandhi Mezhugu, when administered at single dose, is non-toxic and can be used safely as oral formulation. Sub-Acute Toxicity Study There were no signs of toxicity or mortality observed in both sexes of rats treated at 9, 5 and orally for a dosing period of 8 days and in the recovery group of rats and did not show any significant differences in bodyweight between control and treated groups during this period (Figure 1). Haematological parameters such as haemoglobin, red blood cells, white blood cells, mean corpuscular haemoglobin, though found to be statistically significant at low and mid doses as compared to the control, they were found to be well within the normal range of 187
7 rats in experimental groups (Table ). Hence the changes observed were not treatment-related effects. All the biochemical parameters such as glucose, total cholesterol, triglycerides, total protein, alkaline phosphatase, creatinine, blood urea were within the normal range (Table ) observed between control and treated groups. The levels of liver marker enzymes like SGOT and SGPT were found to be well within the normal range for rats in NM treated groups (Table ). In this study, histopathological examinations in control and high dose group revealed no abnormalities. There were no haematological, biochemical and histopathological alterations observed with NM administration even at twice daily in rats for a period of 8 days compared to control. The No Observed Adverse Effect Level (NOAEL) of NM was estimated to be greater than /day in rats. Hence, it is concluded that NM is safe for oral administration in rat up to /day. Sub Chronic Toxicity Study There were no signs of toxicity or mortality observed in both sexes of rats treated at 9, 5 and orally for a period of 90 days (intermittently with 7 days of dosing and 7 days of drug holiday) and in the recovery group of rats and did not show any significant differences in bodyweight between control and treated groups during this period (Figure ). Haematological parameters such as haemoglobin, red blood cells, white blood cells, mean corpuscular haemoglobin were found to be well within the normal range for rats in experimental groups (Table 5). All the parameters in serum biochemical profile such as glucose, total cholesterol, triglycerides, total protein, alkaline phosphatase, creatinine, blood urea were within the normal range (Table 6). The levels of liver marker enzymes like SGOT and SGPT were found to be well within the clinical range for rats in NM treated groups (Table 7). In our study, histopathological examinations in control and high dose group revealed no abnormalities. There were no haematological, biochemical and histopathological alterations observed with NM administration even at twice daily in rats for a period of 90 days compared to control. The No Observed Adverse Effect Level (NOAEL) of NM was estimated to be greater than /day in rats. Hence, it can be concluded that NM is safe for oral administration. ICP-OES Detection of Heavy Metal Traces in Animal Tissue Samples (Brain, Liver, Kidney) in Sub-Chronic Toxicity Study ( group) of Nandhi Mezhugu In sub chronic repeated oral 90 days toxicity study of Nandhi Mezhugu in Wistar rats (OECD 08) the tissues of brain, kidney, and liver of high dose group were subjected to ICP-OES study to screen for heavy metal content and it was found that there was absence of traces of heavy metals such as lead, cadmium, mercury, and arsenic. The toxic effects of mercury were neutralized in the presence of sulphur, which is one of the ingredients in NM 9. From other safety studies on herbo-metallo mineral formulations 9,10, it was evident that if the preparation of such formulations is carried out as per Standard Operating Procedures (they undergo thorough extensive purification and preparation methods involving distillation, calcination, heating, melting and extraction using earthen utensils at specified temperatures to make the minerals ready for human consumption) mentioned in classical literature, they do not produce any toxic effects. In the prepared NM, the essential elements such as Na, K, Ca, Mg, Cu, Fe and Zn had also been found in µg/g amounts and trace amounts of arsenic and mercury seemed to remain chelated with organic ligands derived from medicinal herbs by alchemic processes making these biologically assimilable. If the Siddha herbo-metallo mineral formulation containing biologically-produced nanoparticles is taken along with palm jiggery, honey, ghee, and milk, the latter makes these elements easily assimilable, eliminating their harmful effects and enhancing their biocompatibility. It is well-established that several metals play a vital role in the biochemical processes as well as in the cure of many diseases. It is believed that heavy metals such as Hg and As, which are widely used in the Siddha system of medicine act as catalysts, which exert their catalytic activity by virtue of their presence in the intestines without ever reaching the bloodstream. It is, probably, this property which renders many of the highly toxic metals, nontoxic. It is believed that the toxic effects of these medicines are neutralized by the medium of honey/ghee/milk and provide a natural and effective alternative to synthetic allopathic drugs 9. CONCLUSION At present, the world s focus has turned towards traditional systems of medicine because of the adverse effects reported from other systems of medicine in treating the debilitating diseases. The Siddha system of medicine is very powerful among the Indian systems of medicine. Nandhi mezhugu is a sasthric siddha herbo-metallo mineral formulation, which has gained a place in treating autoimmune diseases like rheumatoid arthritis and different types of cancers etc. The drug NM has been validated scientifically in the area of safety. ACKNOWLEDGEMENT The authors acknowledge Late. Dr. J.Rani MVSc., consultant veterinarian National Institute of Siddha(NIS) for her valuable support during the study period. The authors acknowledge the assistances rendered by Dr. Sathish, Dr. Rathinam and Dr. Harish Anbuselvan, PG scholars Department of Gunapadam, NIS. The authors appreciate the services provided by the animal house attenders of NIS Mr. J.Lingam, Mrs. R.Devi and Mr. Venkateshvaralu. REFERENCES 1. Kuppusamy mudhaliyar KN, Uththamaraayan KS. Siddha vaidhiya thirattu. 1st ed. Chennai (TN): Department of Indian medicine and Homoeopathy.009. p Anonymous The siddha formulary of India. Part 1. Department of ISM & H, Ministry of AYUSH, Health and Family Welfare, Government of India, 1st ed. New Delhi: 199. p Lakshmi kantham T et al. Standardization of Nandhi Mezhugu, a poly herbomineral Siddha formulation. J Pharmacy Research 016; 10(10): Available from: OECD, OECD Guideline for testing of chemicals, Acute oral toxicity-acute toxic class method: TG -Adopted, OECD, Paris, France: OECD, OECD Guideline for testing of chemicals, Repeated dose 8-day oral toxicity study in rodents: TG 07-Adopted, OECD, Paris, France: OECD, OECD Guideline for testing of chemicals, Repeated dose 90-day oral toxicity study in rodents: TG 08-Adopted, OECD, Paris, France: Gulkarni SK. Hand book of experimental pharmacology. rd ed. New Delhi: Vallabh Prakashan Globally harmonized system of classification and labelling of chemicals (GHS). Health and environmental hazards classification criteria for substances and mixtures ppt;015;
8 9. Kumar A, Nair AGC, Reddy AVR, Garg AN. Bhasmas unique ayurvedic metallic herbal preparations, chemical characterization: May 10: Gajendra kumar, Yogendra kumar gupta. Evidence for safety of ayurvedic herbal, herbo-metallic and bhasma preparations on neurobehavioral activity and oxidative stress in rats. 01: An international quarterly J research in Ayurveda. Oct-Dec; (): Cite this article as: Lakshmi Kantham T et al. Acute, sub-acute (8-days), and subchronic (90-days) oral toxicity studies of Nandhi mezhugu. Int. J. Res. Ayurveda Pharm. 017;8(Suppl ): Source of support: Nil, Conflict of interest: None Declared Disclaimer: IJRAP is solely owned by Moksha Publishing House - A non-profit publishing house, dedicated to publish quality research, while every effort has been taken to verify the accuracy of the content published in our Journal. IJRAP cannot accept any responsibility or liability for the site content and articles published. The views expressed in articles by our contributing authors are not necessarily those of IJRAP editor or editorial board members. 189
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